title: Recombinant methionyl bovine prolactin: loss of bioactivity after single amino acid deletions from putative helical regions.
abstract: We have previously described a method for producing recombinant methionyl bovine PRL (Met-bPRL), which is as bioactive as the authentic hormone in the Nb2 cell lactogen bioassay; in contrast, a Met-bPRL variant lacking tyrosine 28 was essentially devoid of bioactivity. In the present study we have investigated this loss of bioactivity at the molecular level by determining the bioactivities of a number of Met-bPRL variants engineered to contain specific changes in their primary structures. It was found that the presence of tyrosine per se at the 28 position in Met-bPRL was not essential for high bioactivity, since Met-bPRL variants prepared by replacing tyrosine 28 with other amino acids (arginine, phenylalanine, alanine, and histidine) still had substantial bioactivity (40-74% that of Met-bPRL). Neither was the loss of bioactivity related to a shift in the relative positions of conserved histidines 27 and 30; in fact, histidine 27 was found not to be essential for the bioactivity of the hormone. The loss of bioactivity after deletion of tyrosine 28 from Met-bPRL appears to be related to the removal of an amino acid from the middle of a putative helix (no. 1) rather than to the loss of a residue specific to lactogen function. This suggestion is supported by the finding that Met-bPRL variants obtained by deletion of selected single amino acids from center domains of putative helix 2, 3, or 4 were also essentially devoid of bioactivity. It is speculated that this lack of bioactivity reflects an inability of the proteins to assume a native conformation.
authors.affiliation: Department of Biochemistry, Faculty of Medicine, University of British Columbia, Vancouver, Canada.
authors.forename: D N
authors.initials: DN
authors.lastname: Luck
authors.forename: P W
authors.initials: PW
authors.lastname: Gout
authors.forename: K
authors.initials: K
authors.lastname: Kelsay
authors.forename: T
authors.initials: T
authors.lastname: Atkinson
authors.forename: C T
authors.initials: CT
authors.lastname: Beer
authors.forename: M
authors.initials: M
authors.lastname: Smith
mesh_terms: D000595:Amino Acid Sequence; D002872:Chromosome Deletion; D015870:Gene Expression; D014644:Genetic Variation; D008969:Molecular Sequence Data; D011388:Prolactin / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000235:genetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; C060530:methionylprolactin; D011388:Prolactin